Previous Article | Next Article 
Molecular and Cellular Biology, July 2000, p. 5227-5234, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
c-Jun NH2-Terminal Kinase Inhibits
Targeting of the Protein Phosphatase Calcineurin to NFATc1
Chi-Wing
Chow,1
Chen
Dong,2
Richard A.
Flavell,2 and
Roger J.
Davis1,*
Howard Hughes Medical Institute, Program in Molecular
Medicine, Department of Biochemistry and Molecular Biology, University
of Massachusetts Medical School, Worcester, Massachusetts
01605,1 and Howard Hughes Medical
Institute, Section of Immunobiology, Yale University School of
Medicine, New Haven, Connecticut 065202
Received 10 January 2000/Returned for modification 24 February
2000/Accepted 12 April 2000
 |
ABSTRACT |
The protein phosphatase calcineurin is a critical mediator of
calcium signals during T-cell activation. One substrate of calcineurin is the transcription factor NFATc1, which is retained in the cytoplasm of quiescent cells. NFATc1 activation requires the translocation of the
transcription factor into the nucleus, a process that is mediated by
calcineurin. This interaction with calcineurin requires a targeting
domain (PxIxIT motif) located in the NH2-terminal region of
NFATc1. Here we demonstrate that the calcineurin targeting domain of
NFATc1 is phosphorylated and inactivated by the c-Jun NH2-terminal kinase (JNK). This disruption of calcineurin
targeting inhibits the nuclear accumulation and transcription activity
of NFATc1 and accounts for the observation that
Jnk1
/ T cells exhibit greatly increased
NFATc1-dependent nuclear responses.
| |
INTRODUCTION |
The c-Jun NH2-terminal
kinase (JNK) group of mitogen-activated protein kinases is activated by
treatment of cells with cytokines or exposure to environmental stress
(19). Gene disruption studies indicate that JNK protein
kinases are required for multiple biological processes (15, 23,
32, 41, 42). However, the molecular mechanisms that account for
the phenotypes displayed by JNK knockout mice remain unclear. For
example, the disruption of the Jnk1 gene causes a severe
defect in the response of CD4+ helper T (TH)
cells to antigen-presenting cells (15). Wild-type naive
TH cells can differentiate to TH1 or
TH2 effector cells, which mediate inflammatory and humoral
responses, respectively. In contrast, Jnk1/
CD4 TH cells preferentially differentiate to
TH2 effector cells and secrete large amounts of the
TH2 cytokines, including interleukin-4 (IL-4). As a
consequence, these mice are highly susceptible to infection with
Leishmania. These effects of Jnk1 gene disruption were associated with increased nuclear accumulation of the NFATc1 transcription factor (15), which is known to act at the IL-4 promoter and is essential for TH2 responses (30,
44). The phenotype of Jnk1/ mice
suggests that NFATc1 is negatively regulated by JNK1 (15). However, this phenotype could be a direct or an indirect consequence of
Jnk1 gene disruption.
Transcription factor NFAT was first identified as an important
regulator of IL-2 gene expression (16, 20). More recently, it has been established that NFAT contributes to the expression of
several cytokines and participates in multiple physiological processes
(12, 31). In resting T cells, NFAT is restricted to the
cytoplasm (12, 31). Following T-cell activation, a sustained
increase in intracellular calcium (37) activates the phosphatase calcineurin (11, 21). The activated calcineurin dephosphorylates NFAT and leads to increased nuclear accumulation (12, 31). The nuclear NFAT transcription factor then
increases the expression of target genes, including IL-2, IL-4, and Fas ligand.
The interaction of calcineurin with NFAT is a critical element in the
signal transduction pathway that leads to increased NFAT-dependent gene
expression. Interestingly, this interaction is mediated by a targeting
domain (PxIxIT motif) that is present in the NH2-terminal
region of the NFAT transcription factor (1, 2, 8). This
targeting domain is required for efficient NFAT activation in vivo.
Furthermore, ectopic expression of the targeting domain causes profound
and specific inhibition of NFAT-mediated gene expression in cultured
cells (2, 8). Studies of transgenic mice also demonstrate
inhibition of NFAT-mediated gene expression caused by expression of the
calcineurin targeting domain (8). Together, these data
indicate that the calcineurin targeting domain is a critical component
of the regulatory mechanism that controls NFAT activity.
The purpose of this study was to examine the role of JNK1 in NFATc1
regulation. Since the phenotype of Jnk1/
mice suggests a correlation between the JNK1 signaling pathway and
NFATc1 (15), we tested whether NFATc1 is directly regulated by JNK1. We report that JNK1 binds and phosphorylates NFATc1 on sites
located near the PxIxIT calcineurin targeting domain. This phosphorylation inhibits the interaction of calcineurin with the targeting domain and blocks nuclear accumulation. This observation provides a molecular mechanism for the observation of increased NFATc1
nuclear accumulation in Jnk1/ mice.
| |
MATERIALS AND METHODS |
Reagents.
The IL-4 luciferase reporter plasmid
(36) and the expression vectors for calcineurin
(28), NFATc1 (18), NFATc1-
(25), dominant negative NFAT (dnNFAT) (8), and JNK signaling
pathway components (7, 40) have been described elsewhere.
The NFATc1-
expression vector was constructed by cloning a
PCR-derived cDNA in the BamHI site of pSR. Recombinant
NFAT proteins were expressed using the vector pGEX-5X1
(Amersham-Pharmacia Biotech) by cloning cDNA fragments in the
BamHI and NotI sites. Deletion and point mutations were constructed by PCR and sequenced with an Applied Biosystems machine. Bacterially expressed CRM1 was purified by glutathione (GSH) affinity chromatography (43). The
phosphospecific NFATc1 antibody prepared by immunization of rabbits
with ovalbumin conjugated with glutaraldehyde to the synthetic
phosphopeptide Ala-Pro-Ala-Leu-Glu-Ser(P)-Pro-Arg-Ile-Glu-Ile-Thr-Ser-Cys-Leu. The phosphospecific NFATc1 antibody was affinity purified from the
rabbit serum using standard techniques (17).
Binding assays.
Cell extracts prepared using Triton-lysis
buffer (20 mM Tris [pH 7.4], 137 mM NaCl, 2 mM EDTA, 1% Triton
X-100, 25 mM -glycerophosphate, 1 mM sodium vanadate, 2 mM sodium
pyrophosphate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin per ml) were incubated (5 h at 4°C) with
recombinant proteins (5 µg) prebound to 20 µl of GSH-Sepharose.
After three washes with Triton-lysis buffer, the bound proteins were
detected by protein immunoblot analysis.
Kinase assays.
Hemagglutinin epitope (HA)-tagged JNK1 was
coexpressed together with and without MLK3 in COS cells. Cell extracts
were prepared with Triton-lysis buffer 48 h after transfection.
JNK1 immunocomplex kinase assays were performed with recombinant NFATc1
proteins (1 µg) as the substrate (7).
Immunofluorescence assays.
BHK cells were transfected using
Lipofectamine (Life-Technologies Inc.) according to the manufacturer's
protocol. Immunofluorescence analysis was performed using cells
transfected with an NFATc1- expression vector (1 µg) together with
and without expression vectors for activated calcineurin (0.2 µg),
MKK7 (0.2 µg) plus JNK1 (0.2 µg), dnJNK1 (0.5 µg), or JIP-1 (0.3 and 0.7 µg). NFATc1 proteins were detected using monoclonal antibody
7A6 (1:200; Affinity Bioreagents). The secondary antibody was Texas
red-conjugated anti-mouse immunoglobulin antibody (1:100; Jackson
ImmunoResearch). Nuclei were visualized using
4',6-diamidino-2-phenylindole (Sigma).
| |
RESULTS |
JNK inhibits the nuclear accumulation of NFATc1-.
To test
whether JNK1 regulates NFATc1, we examined the effect of activated JNK1
in transfection assays using cultured Jurkat T cells. Treatment with
phorbol ester and ionomycin caused a transient increase in JNK protein
kinase activity which returned to basal levels within 1 h
(24, 35). At later times, a large increase in the
expression of NFATc1 was detected (Fig.
1A). Cell fractionation studies
demonstrated the presence of NFATc1 in the nucleus. Expression of
activated JNK1 did not alter the expression of NFATc1 but did cause a
marked reduction in the amount of nuclear NFATc1 (Fig. 1A). In
contrast, expression of dnJNK1 increased the amount of nuclear NFATc1 caused by anti-CD3 stimulation (data not shown). Transfection assays were performed to examine the effect of JNK1 on
transcription activity using an IL-4 promoter reporter gene. Activated
JNK1, like dnNFAT, strongly inhibited reporter gene expression (Fig.
1B). Since constitutively nuclear mutant NFATc1 caused greatly
increased reporter gene expression (data not shown), it is likely that
the effect of activated JNK1 to inhibit reporter gene expression (Fig.
1B) was the consequence of the decreased amount of nuclear NFATc1 (Fig.
1A). Together, these data demonstrate that JNK1 functions as an
inhibitor of the endogenous NFATc1 transcription factor expressed by
Jurkat T cells.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 1.
JNK1 inhibits NFAT activity in T cells. (A) Activation
of JNK1 inhibits NFATc1 nuclear localization. Jurkat T cells were
transfected without (empty expression vector; Control) and with MKK7
plus JNK1 (JNK). After 16 h, the cells were stimulated without
( ) and with (+) ionomycin (I; 2 µM) plus PMA (P; 100 nM) for 6 h. Nuclear extracts were isolated (14), and NFATc1 was
examined by protein immunoblot analysis using monoclonal antibody 7A6
(Affinity Bioreagents). Similar data were obtained in three independent
experiments. (B) Activated JNK inhibits NFAT transcription activity.
Jurkat T cells were transfected with an IL-4 promoter reporter gene
(firefly luciferase) plasmid together without (Control) and with MKK7
plus JNK1 (JNK) or dnNFAT. Transfection efficiency was monitored by
measurement of Renilla luciferase activity. The cells were
stimulated without ( ) and with ( ) ionomycin (2 µM) plus PMA
(100 nM) for 6 h. The data are presented as fold activation by in
treated compared to untreated cells (mean ± standard deviation;
n = 4).
|
|
Several alternatively spliced variant forms of NFATc1 have been
described (
9,
10,
18,
25,
26,
33). The major
isoform
expressed by T cells has been identified as NFATc1-
(
25,
33). We therefore performed further analysis of the
regulation
of NFATc1 by JNK using the NFATc1-
isoform.
JNK phosphorylates NFATc1-.
The JNK1 protein kinase binds
and phosphorylates its substrates (19). Deletion analysis
and in vitro binding assays indicated that NFATc1- residues 126 to
138 are required for the binding of NFATc1- to JNK1 (Fig.
2). In contrast, NFATc1-
phosphorylation by JNK1 was reduced by truncation at residue 171 and
was eliminated by truncation at residues 126 (Fig.
3A). Phosphoamino acid analysis demonstrated the presence of phosphoserine (data not shown). Four potential JNK1 phosphorylation sites (Ser-Pro motifs) were identified. Mutational analysis indicated that both Ser117 and
Ser172 were phosphorylated by JNK1. Replacement of either
Ser117 or Ser172 with Ala reduced
phosphorylation by JNK1, while the replacement of both
Ser117 and Ser172 eliminated phosphorylation by
JNK1 (Fig. 3B). Together, these data demonstrate that JNK1 binds
NFATc1- and phosphorylates both Ser117 and
Ser172 in vitro. The JNK1 phosphorylation site
Ser172, but not Ser117, is conserved in the
related transcription factor NFATc3 (7). Comparative
tryptic phosphopeptide mapping of NFATc1- isolated from
[32P]phosphate-labeled cells indicated that two
phosphopeptides present in maps of wild-type NFATc1- were absent in
maps of mutated [Ala117 Ala172]
NFATc1-, suggesting that Ser117 and Ser172
may be phosphorylated in vivo (data not shown). To test this hypothesis, we prepared a phospho-NFATc1 antibody and performed immunoblot analysis of NFATc-. The phospho-NFATc1 antibody
specifically detected phosphorylation on Ser117 (Fig.
4A). Activation of endogenous JNK
caused increased NFATc1- phosphorylation (Fig. 4B).
Overexpression of the scaffold protein JIP-1 causes profound inhibition
of JNK (13, 39) and inhibited NFATc1- phosphorylation
(Fig. 4B). Together, these data suggest that NFATc1- is a JNK
substrate in vivo.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 2.
Interaction of JNK1 with NFATc1-. Lysates were
prepared from COS cells transfected with HA-JNK1, and the binding of
HA-JNK1 to immobilized recombinant NFATc1- was examined. The effect
NFATc1- deletions is shown. The location of the JNK binding domain
(JBD) and PxIxIT motif on NFATc1- are illustrated schematically.
Potential JNK phosphorylation sites (SP) are indicated.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 3.
Phosphorylation of NFATc1- by JNK1 in vitro. (A) JNK1
phosphorylates NFATc1-. Immunocomplex kinase assays were performed
using JNK1 activated without (Control; ) or with (+) MLK3 using
recombinant NFATc1- as the substrate. Phosphorylated NFATc1- was
detected by autoradiography and quantitated by PhosphorImager
(Molecular Dynamics) analysis. (B) JNK phosphorylates NFATc1- on
Ser117 and Ser172. Wild-type and mutant
NFATc1- proteins (residues 1 to 202) were phosphorylated by JNK1 in
vitro. The effects of JNK1 activation by MLK3 and the replacement of
Ser117 and Ser172 with Ala were examined.
|
|
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 4.
Phosphorylation of NFATc1- in vivo. (A) NFATc1-
was coexpressed with MKK7 plus JNK1 in COS cells. The effect of
replacement of Ser117 and Ser172 with Ala
residues was investigated. Cell lysates were examined by protein
immunoblot analysis by sequentially probing with
anti-phospho-NFATc1- and anti-NFATc1. A nonspecific band
was detected by the phospho-NFATc1- antibody immediately above the
NFATc1- band. (B) NFATc1- was coexpressed without () and with
(+) the JNK inhibitor JIP-1 in COS cells. The cells were treated
without () and with (+) UV-C radiation (80 J/m2) and
harvested after 30 min. The effect of replacement of the JNK
phosphorylation sites (Ser117 and Ser172) with
Ala was examined. The NFATc1- proteins were detected by protein
immunoblot analysis by sequentially probing with
anti-phospho-NFATc1- and anti-NFATc1.
|
|
Phosphorylation by JNK inhibits the nuclear accumulation of
NFATc1-.
Studies of Jurkat T cells demonstrated that activated
JNK1 inhibited the calcium-stimulated nuclear accumulation of NFATc1 (Fig. 1A). This observation was confirmed by immunofluorescence analysis of the subcellular distribution of NFATc1-. Activated JNK1 inhibited the calcineurin-stimulated nuclear accumulation of
NFATc1- (Fig. 5A and B). This effect
of activated JNK1 to inhibit NFATc1- nuclear accumulation was
blocked by overexpression of proteins that can inhibit JNK signaling,
including dnJNK1 (Figure 5C) and the scaffold protein JIP-1 (Fig. 5D).
Together, these data demonstrate that activated JNK1 regulates
NFATc1- nuclear accumulation.
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 5.
JNK1 inhibits the nuclear accumulation of NFATc1-. (A
and B) Subcellular distribution of NFATc1-, examined by
immunofluorescence analysis in transfected BHK cells. The NFAT proteins
(red; right) and nucleus (blue; left) were visualized (A). The effects
of expression of a constitutively activated calcineurin ( CN) and
MKK7 plus JNK1 (JNK) were examined. Arrowheads indicate the nuclei of
cells expressing transfected proteins. The data were quantitated (B)
following examination of 300 transfected cells and are presented as the
percentage of cells with nuclear NFATc1- (mean ± standard
deviation [SD]; n = 3). (C) Effect of dnJNK on the
subcellular distribution of NFATc1-, examined by immunofluorescence
analysis. The effects of expression of constitutively activated
calcineurin (CN) and MKK7 plus JNK1 (JNK) were examined. The
percentage of cells with nuclear NFATc1- is presented (mean ± SD; n = 3). (D) Effect of JIP-1 on the subcellular
distribution of NFATc1-, examined by immunofluorescence analysis.
The percentage of cells with nuclear NFATc1- is presented (mean ± SD; n = 3). The effects of increasing amounts of
JIP-1 expression vector (0.3 and 0.7 µg) and the expression of
constitutively activated calcineurin (CN) and MKK7 plus JNK1 (JNK)
were examined. (E) Mutational removal of Ser117 and
Ser172 potentiates NFATc1- transcription activity on the
IL-4 promoter. Wild-type and [Ala117 Ala172]
NFATc1- were cotransfected with an IL-4 reporter plasmid in the
absence (Untreated) and presence of MKK7 plus JNK1 (JNK). The cells
were stimulated without (Untreated) and with PMA (P; 100 nM) plus
ionomycin (I; 2 µM) for 16 h. The data are presented as fold
activation compared to an untreated control (cells transfected without
an NFATc1- expression vector).
|
|
To test whether the phosphorylation of NFATc1-
is mechanistically
relevant to the regulation of NFATc1-
nuclear accumulation
by JNK1,
we examined the effect of the replacement of the NFATc1-
phosphorylation sites with Ala residues. The subcellular distribution
of the wild-type and mutated NFATc1-
proteins was examined by
immunofluorescence analysis. Mutations at both phosphorylation
sites
(Ser
117 and Ser
172) altered the subcellular
distribution of NFATc1-
. The mutated
[Ala
117
Ala
172] NFATc1-
protein was found to be present in
the nucleus under
basal conditions and was not regulated by
activated JNK1 (Fig.
5A and B). Mutation at either Ser
117
or Ser
172 was sufficient to increase the nuclear
accumulation of NFATc1-
.
To examine whether the altered nuclear
accumulation of the phosphorylation-defective
NFATc1-
proteins was
relevant to transcription activity, we performed
transfection assays of
Jurkat T cells with an IL-4 promoter reporter
plasmid (Fig.
5E).
Expression of wild-type NFATc1-
using Jurkat
T cells caused a large
increase in phorbol myristate acetate (PMA)-ionomycin-stimulated
IL-4
promoter reporter gene expression, which was inhibited by
coexpression
of activated JNK1. In contrast, the [Ala
117
Ala
172] NFATc1-
protein caused increased reporter gene
expression in
the absence of PMA-ionomycin stimulation and was not
inhibited
by activated JNK1 in the presence or absence of PMA-ionomycin
(Fig.
5E). Together, these data demonstrate that the JNK1
phosphorylation
sites (Ser
117 and Ser
172) are
required for the regulation of both nuclear accumulation
and
transcription activity of NFATc1-
by
JNK1.
JNK selectively regulates NFATc1 isoforms.
We have previously
reported that recombinant NFATc1 is not regulated by JNK1
(7). This conclusion markedly differs from the results
obtained from the analysis of endogenous NFATc1 expressed by Jurkat T
cells (Fig. 1). Several alternatively spliced variant forms of NFATc1
have been described, including molecules with three distinct
COOH-terminal domains (9, 10) and three distinct NH2-terminal domains (18, 25, 26, 33). The
isoforms with distinct NH2-terminal domains correspond to
NFATc1 (18), NFATc1- (25), and
NFATc1- (26, 33). NFATc1- is the major isoform expressed by T cells (25, 33).
To test whether JNK may differentially interact with NFATc1
isoforms, we expressed the NH
2-terminal region of NFATc1,
NFATc1-
,
and NFATc1-
in bacteria as glutathione
S-transferase (GST) fusion
proteins. The purified GST-NFAT
proteins were immobilized on GSH-agarose.
Control experiments
demonstrated that JNK1 did not bind to immobilized
GST
(Fig.
6A). However, strong binding of
JNK1 to NFATc1-
and
NFATc1-
was detected. In contrast, NFATc1
bound poorly to JNK1.
In vitro immune complex protein kinase
assays using epitope-tagged
JNK1 demonstrated that there was
greater phosphorylation of NFATc1-
and NFATc1-
than of
NFATc1 (Fig.
6B). These data suggest that
JNK1 may be selectively
targeted to NFATc1-
and NFATc1-
.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 6.
Differential regulation of NFATc1 isoforms by JNK. (A)
The NH2-terminal regions of NFATc1 (18),
NFATc1- (25), and NFATc1- (26, 33) are
illustrated schematically. The PxIxIT motif (hatched box), the NFAT
homology domain (NHD), and the alternative NFATc1 NH2
termini are indicated. Immobilized GST, GST-NFATc1 (residues 1 to 126),
GST-NFATc1- (residues 1 to 202), and GST-NFATc1- (residues 1 to
189) was incubated with extracts prepared from COS cells transfected
with HA-JNK1. The binding of HA-JNK1 was examined by immunoblot
analysis. (B) Comparison of the phosphorylation of NFATc1 isoforms by
JNK1 in vitro. Immunocomplex kinase assays were performed using Flag
epitope-tagged JNK1 activated without () and with (+) MLK3, using
recombinant NFATc1 isoforms as the substrate. (C and D) Subcellular
distribution of NFATc1 isoforms, examined by immunofluorescence
analysis in transfected BHK cells. The NFAT proteins (red; right) and
nucleus (blue; left) were visualized (C). The effect of constitutively
activated calcineurin (CN) or MKK7 plus JNK1 (JNK) was examined.
Arrowheads indicate the nuclei of cells expressing transfected
proteins. The data were quantitated (D) following examination of 300 transfected cells and are presented as the percentage of cells with
nuclear NFATc1 (mean ± standard deviation; n = 3).
|
|
We compared the subcellular distribution of NFATc1, NFATc1-
, and
NFATc1-
by immunofluorescence analysis (Fig.
6C). In control
cells,
NFATc1-
and NFATc1-
were located predominantly in the
cytoplasm,
but a large fraction of NFATc1 was found in the nucleus.
Expression of
activated JNK1 caused no significant change in the
subcellular
distribution of these NFATc1 isoforms. In contrast,
activated
calcineurin caused increased nuclear localization of
all
three NFATc1 isoforms. Activated JNK1 suppressed the
calcineurin-stimulated
nuclear accumulation of NFATc1-
and
NFATc1-
but not the NFATc1
isoform.
Taken together, these data indicate that JNK1 negatively
regulates the NFATc1-
and NFATc1-
isoforms but not NFATc1.
It is
likely that the markedly decreased binding of JNK1 to
NFATc1 (Fig.
6A) compared to the other NFATc1 isoforms contributes to
this
differential regulation. As NFATc1-
is the major isoform
expressed
by T cells (
25,
33), these data indicate that JNK1
may function
as an inhibitor in T cells. The phenotype of
Jnk1/ mice is consistent with the hypothesis
(
15).
Phosphorylation by JNK inhibits targeting of NFATc1- by the
phosphatase calcineurin.
JNK1 phosphorylates NFATc1- on
Ser117 and Ser172 (Fig. 3). Ser117
of NFATc1- is located adjacent to the previously identified
calcineurin targeting domain (PxIxIT motif) (1, 2, 8). We
hypothesized that phosphorylation on Ser117 may regulate
the targeting of NFATc1- to calcineurin. However, the proximity of
Ser117 to the calcineurin targeting domain suggests that
Ser117 may represent a potential substrate for
dephosphorylation by calcineurin. We therefore examined the kinetics of
calcineurin-mediated dephosphorylation of JNK1-phosphorylated
NFATc1-. This analysis demonstrated that Ser172 (and not
Ser117) was preferentially dephosphorylated by calcineurin
in vitro (Fig. 7A). Thus, the PxIxIT
targeting motif that binds calcineurin facilitates the
dephosphorylation of distal (e.g., Ser172) but not proximal
(e.g., Ser117) NFAT phosphorylation sites. These data
support the hypothesis that Ser117 may regulate the
targeting function of the NFATc1- PxIxIT motif. To test this
hypothesis, we examined the effect of NFATc1- phosphorylation by
JNK1 on the interaction of NFATc1- with calcineurin. Phosphorylation caused a marked reduction in the binding of calcineurin to NFATc1- (Fig. 7B). Mutational analysis demonstrated that the phosphorylation of
Ser117, but not Ser172, was required for the
regulation of calcineurin binding by JNK1. To confirm that
phosphorylation on Ser117 regulates calcineurin binding, we
performed competition assays using a synthetic PxIxIT peptide with
either Ser117 or phospho-Ser117 (Fig. 7C).
Binding assays confirmed that the Ser117 PxIxIT peptide
disrupted calcineurin binding in a dose-dependent manner. In contrast,
calcineurin binding to NFATc1- was not inhibited by the
phospho-Ser117 PxIxIT synthetic peptide. Together, these
data demonstrate that Ser117 phosphorylation inhibits the
function of the calcineurin targeting domain of NFATc1-.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 7.
JNK1 inhibits the binding of calcineurin to NFATc1-.
(A) Comparison of calcineurin-stimulated dephosphorylion of
Ser117 and Ser172. Recombinant NFATc1-
(residues 1 to 202) was phosphorylated by JNK1 in vitro and incubated
(30 min at 30°C) in 50 mM HEPES (pH 7.4)-2 mM MnCl2-0.5
mM EDTA-15 mM 2-mercaptoethanol-0.1 mg of bovine serum albumin per ml
together with calmodulin (250 U) and indicated amounts of calcineurin
(Sigma). The phosphorylated NFATc1- was detected after sodium
dodecyl sulfate-polyacrylamide gel electrophoresis by autoradiography
and was quantitated by PhosphorImager (Molecular Dynamics) analysis.
Dephosphorylation at Ser117 and Ser172 was
examined using [Ala172] and [Ala117]
NFATc1-, respectively. (B) Phosphorylation of NFATc1- by JNK1
inhibits the binding of NFATc1- to the phosphatase calcineurin.
Recombinant NFATc1- (residues 1 to 202) was phosphorylated by JNK1
in vitro. The NFATc1- proteins were immobilized on GSH-Sepharose,
incubated with cell extracts, and washed; bound calcineurin was
detected by protein immunoblot analysis. The effect of replacement of
the NFATc1- phosphorylation sites (Ser117 and
Ser172) with Ala was examined. (C) Phosphorylation of
Ser117 inhibits calcineurin binding to NFATc1-.
Immobilized recombinant NFATc1- (residues 1 to 202) was incubated
with cell extracts, and the binding of calcineurin was examined by
protein immunoblot analysis. Competition analysis was performed by
investigating the effects of various concentrations (0, 0.7, 1.4, 7, and 14 µM) of a synthetic peptide corresponding to the PxIxIT motif
which mediates the targeting of calcineurin to NFATc1-. The effect
of the phosphorylation of the synthetic peptide on Ser117
was examined.
|
|
CRM1 interacts with dephosphorylated NFATc1-.
The export of
NFAT from the nucleus is mediated, in part, by a mechanism that
involves the GTPase Ran and the exportin CRM1 (22). One CRM1
binding site on NFATc3 is located adjacent to the calcineurin targeting
domain (45). Indeed, binding of NFATc3 to calcineurin
competes with the binding to CRM1 (45). Since NFATc1-
phosphorylation inhibits the binding of calcineurin (Fig. 7), we
examined whether phosphorylation affected the interaction of NFATc1-
with CRM1. Binding assays were performed using immobilized GST-CRM1 and
lysates prepared from COS cells expressing NFATc1- (Fig.
8). We found that NFATc1- bound to
CRM1. The binding to CRM1 was increased when the NFATc1- was
coexpressed with activated calcineurin (Fig. 8A). This observation
suggests that calcineurin-mediated dephosphorylation of NFATc1-
increases binding to CRM1.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 8.
NFATc1- binding to CRM1 is regulated by
phosphorylation. (A) Activated calcineurin increases the binding of
NFATc1- to CRM1. NFATc1- was expressed without () or with (+)
activated calcineurin (CN) in COS cells. Recombinant CRM1 was
immobilized on GSH-Sepharose, incubated with the COS cell extracts, and
washed; bound NFATc1- was detected by protein immunoblot analysis.
(B) JNK phosphorylation inhibits the binding of NFATc1- to CRM1. The
effect of replacement of the JNK phosphorylation sites
(Ser117 and Ser172) on the binding of
NFATc1- to CRM1 was examined. The effect of JNK inhibition was
investigated by overexpression of the scaffold protein JIP-1.
|
|
To test whether phosphorylation of NFATc1-
on the JNK sites altered
CRM1 binding, we examined the effect of replacement of
Ser
117 and Ser
172 with Ala residues. A marked
increase in NFATc1-
binding to CRM1
was detected for the mutated,
phosphorylation-defective NFATc1-
protein (Fig.
8B). These data
suggested that phosphorylation on
Ser
117 and
Ser
172 may inhibit binding to CRM1. To test this
hypothesis, we examined
the effect of inhibiting JNK signaling on
the binding of NFATc1-
to CRM1. Overexpression of the scaffold
protein JIP-1 causes a
profound inhibition of JNK signaling
(
13,
39). JIP-1 increased
the binding of CRM1 to
wild-type [Ser
117 and Ser
172] NFATc1-
but
not to the mutated phosphorylation-defective [Ala
117 and
Ala
172] NFATc1-
. These data indicate that
phosphorylation regulates
the interaction between CRM1 and
NFATc1-
.
| |
DISCUSSION |
In this study we demonstrate that the JNK signaling pathway plays
a key role as a negative regulator of substrate targeting by the
phosphatase calcineurin. The phosphorylation of NFATc1 by JNK provides
a mechanism whereby NFATc1-driven responses can be terminated, which
can set a threshold for a given response. This is likely to be
important for normal cellular physiology. Indeed, we have demonstrated
that Jnk1/ mice displayed enhanced
TH2 responses due to increased nuclear localization of
NFATc1 (15). Conversely, activation of the JNK1 signaling
pathway inhibits IL-4 gene promoter activity (Fig. 1). Thus, JNK1
functions in TH2 cells to suppress the expression of cytokines that enhance TH2 responses (e.g., IL-4) which
would otherwise lead to the generation of an unbalanced TH
cell immune response to pathogens. Together, the genetic evidence and
biochemical analysis that we present strongly support the hypothesis
that JNK is a physiologically relevant regulator of NFATc1 function.
Recent studies have established that the formation of protein complexes
is a critical aspect of signal transduction mechanisms that ensures
both efficiency and specificity in vivo (27, 40). An example
of the formation of signaling complexes is the requirement of targeting
domains for the interaction of protein kinases and protein phosphatases
with their substrates. We demonstrate that such interactions are
subjected to regulation by phosphorylation. Our results indicate that
phosphorylation of NFATc1 by JNK inhibits targeting of the
phosphatase calcineurin. Since calcineurin functions to induce the
nuclear accumulation of NFAT, phosphorylation by JNK inhibits
NFATc1 activation. Inhibition of phosphatase targeting is a novel
mechanism of regulation by a mitogen activated protein kinase. However,
this regulatory mechanism may also be applicable to other signaling
systems that are regulated by phosphorylation.
Our study further establishes the importance of the PxIxIT motif in the
regulation of calcineurin targeting to NFAT. Previous studies
demonstrated that the PxIxIT motif was required for efficient activation of NFAT by calcineurin (1) and that ectopic
expression of the PxIxIT motif inhibits NFAT-mediated gene expression
in both cultured cells (2, 8) and transgenic mice
(8). Here we demonstrate that the function of the
PxIxIT targeting motif can be regulated by phosphorylation. Our
data suggest that regulated substrate targeting represents a potential
approach for the design of novel therapeutic agents.
Coordination of NFAT binding to calcineurin and CRM1.
It is
established that both the phosphatase calcineurin and the exportin CRM1
bind to NFAT (22, 45). Studies of NFATc3 indicate that one
of the binding sites for CRM1 is located adjacent to the calcineurin
binding site (45). Binding to calcineurin is calcium
dependent and competes with the binding to CRM1. The binding affinity
of NFATc3 for calcineurin/Ca2+ is higher than that for CRM1
(45). These observations have led to the conclusion that
calcineurin acts, in part, to induce nuclear accumulation of NFATc3 by
suppressing the CRM1-dependent export pathway (45). It is
likely that other NFAT proteins are regulated by similar mechanisms.
Our studies of NFATc1-
indicate that phosphorylation by JNK causes
inhibition of calcineurin binding (Fig.
7). This inhibited
interaction
with calcineurin may account for the effect of activated
JNK to prevent
calcium-mediated nuclear accumulation of NFATc1-
(Fig.
5). Since
calcineurin and CRM1 compete for binding to NFAT,
we considered that
the effect of JNK to decrease calcineurin binding
might lead to
increased CRM1 binding. This potential mechanism
suggests that
increased CRM1-mediated export of NFATc1-
from
the nucleus may
contribute to the effect of JNK signaling to inhibit
nuclear
accumulation of NFATc1-
. However, measurement of CRM1
binding
demonstrated that JNK phosphorylation inhibited the binding
of both
CRM1 and calcineurin (Fig.
7 and
8). These data suggest
that the
primary action of JNK to prevent the nuclear accumulation
of NFATc1-
is inhibition of calcineurin
binding.
Regulation of NFAT subcellular distribution by
phosphorylation.
The NFAT transcription factors are
phosphoproteins that are located in the cytosol of resting cells. Upon
dephosphorylation by the phosphatase calcineurin, NFAT accumulates in
the nucleus (12, 31). Conversely, phosphorylation of NFAT
isoforms opposes nuclear accumulation. NFAT phosphorylation has been
shown to be mediated by many different protein kinases (3, 5-7,
29, 34, 46). The relative importance and physiological
significance of each of the protein kinases remains to be established.
The role of NFAT phosphorylation is unclear, but it is likely that
different sites of phosphorylation will regulate different
biological
functions. Here we report that the phosphorylation
of NFATc1-
by JNK
causes inhibition of calcineurin targeting
to the PxIxIT motif (Fig.
7). Previous studies indicate that phosphorylation
on the Ser-rich
region facilitates intramolecular interaction
with the COOH-terminal
nuclear localization sequence (NLS2) of
NFATc1 (
4).
Phosphorylation-dependent intramolecular interaction
within the NFAT
homology region has also been proposed to regulate
NFATc3 subcellular
distribution (
46). Furthermore, NFAT phosphorylation
facilitates intermolecular interaction with 14-3-3 adapter
proteins
(
6). Binding to 14-3-3 masks the
NH
2-terminal NLS1 and may
regulate NFAT subcellular
distribution. These data indicate that
phosphorylation regulates
multiple NFAT intermolecular and intramolecular
interactions that
contribute to the shuttling of NFAT isoforms
between the
nuclear and cytoplasmic compartments of the cell.
Further studies of
NFAT phosphorylation are required before we
can obtain a complete
understanding of the mechanism of regulation
of NFAT nuclear
accumulation.
Role of JNK-regulated NFATc1- activity.
NFATc1- and
NFATc2 represent the major NFAT isoforms expressed by T cells.
NFATc1- (Fig. 1), but not NFATc2 (7), is negatively regulated by JNK. This observation raises an important question concerning the physiological function of JNK as an NFAT inhibitor. Studies of primary naive peripheral T cells indicate that NFATc1- is
expressed at very low levels, but the expression of NFATc1- is
induced following T-cell activation (20, 33). In contrast, NFATc2 is expressed at high levels both before and after T-cell activation (20). This pattern of expression suggests that
JNK is not a significant regulator of NFAT activity in naive T cells or
in T cells at early times following activation. However, a role for JNK
as an inhibitor of NFAT activity at late times following T-cell
activation is implicated.
The possible role for JNK as an NFAT inhibitor at late times following
T-cell activation is consistent with the results of
a recent report
that examined JNK expression by T cells (
38).
JNK1 and JNK2
were found to be expressed at very low levels in
naive peripheral
CD4
+ T cells. Similarly, the JNK activators MKK4 and MKK7
were found
to be expressed at low levels. Activation mediated by the
T-cell
receptor plus the CD28 coreceptor caused a marked induction of
mRNA and protein expression of JNK1, JNK2, MKK4, and MKK7 that
peaked
at approximately 24 h following T-cell activation. The
peak of
expression correlated with increased JNK activity in the
activated T
cells. Together, these data indicate that the JNK
signaling pathway in
T cells is induced during T-cell activation.
This temporal pattern of
expression of the JNK signaling pathway
components is similar to that
of NFATc1-
. The extremely low level
of expression of NFATc1-
and
JNK pathway components in naive
peripheral CD4
+ T cells
strongly argues against a role for JNK during the early
phase of T-cell
activation.
The considerations outlined above indicate that the function of JNK to
inhibit NFATc1-
most likely occurs at late times following
T-cell activation (e.g., 24 h). We propose that JNK functions
to
provide a tonic inhibitory signal that opposes NFATc1-
activation
and expression of downstream target genes (e.g., IL-4). JNK
may
therefore provide a threshold for the maintenance of T-cell
responses
by requiring higher levels of stimulatory signals. This
mechanism
is consistent with the observation that
Jnk1/ CD4
+ T cells are
hyperresponsive to stimulatory signals, including
enhanced production
of IL-4 (
15). This role of JNK in setting
a threshold for
T-cell activation may contribute to tolerance
and the initiation of an
immune
response.
| |
ACKNOWLEDGMENTS |
The first two authors contributed equally to this work.
We thank G. R. Crabtree, J. S. Gutkind, T. Hoey, S. Kornbluth, K. M. Murphy, and T. Soderling for providing reagents;
C. Turck for peptide synthesis; T. Barrett, A. Quail, and J. Stein for technical assistance; and K. Gemme for administrative assistance.
C.-W. Chow is an Arthritis Foundation fellow. This work was supported
in part by grants CA65861 and CA72009 and by the Howard Hughes Medical Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Howard
Hughes Medical Institute Program in Molecular Medicine University
of Massachusetts Medical School, 373 Plantation St., Worcester, MA
01605. Phone: (508) 856-6054. Fax: (508) 856-3210. E-mail:
Roger.Davis{at}Umassmed.Edu.
| |
REFERENCES |
| 1.
|
Aramburu, J.,
F. Garcia-Cozar,
A. Raghavan,
H. Okamura,
A. Rao, and P. G. Hogan.
1998.
Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT.
Mol. Cell
1:627-637[CrossRef][Medline].
|
| 2.
|
Aramburu, J.,
M. B. Yaffe,
C. Lopez-Rodriguez,
L. C. Cantley,
P. G. Hogan, and A. Rao.
1999.
Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A.
Science
285:2129-2133[Abstract/Free Full Text].
|
| 3.
|
Avots, A.,
M. Buttmann,
S. Chuvpilo,
C. Escher,
U. Smola,
A. J. Bannister,
U. R. Rapp,
T. Kouzarides, and E. Serfling.
1999.
CBP/p300 integrates Raf/Rac-signaling pathways in the transcriptional induction of NF-ATc during T cell activation.
Immunity
10:515-524[CrossRef][Medline].
|
| 4.
|
Beals, C. R.,
N. A. Clipstone,
S. N. Ho, and G. R. Crabtree.
1997.
Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction.
Genes Dev.
11:824-834[Abstract/Free Full Text].
|
| 5.
|
Beals, C. R.,
C. M. Sheridan,
C. W. Turck,
P. Gardner, and G. R. Crabtree.
1997.
Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3.
Science
275:1930-1934[Abstract/Free Full Text].
|
| 6.
|
Chow, C.-W., and R. J. Davis.
2000.
Integration of calcium and cyclic AMP signaling pathways by 14-3-3.
Mol. Cell. Biol.
20:702-712[Abstract/Free Full Text].
|
| 7.
|
Chow, C. W.,
M. Rincon,
J. Cavanagh,
M. Dickens, and R. J. Davis.
1997.
Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway.
Science
278:1638-1641[Abstract/Free Full Text].
|
| 8.
|
Chow, C. W.,
M. Rincon, and R. J. Davis.
1999.
Requirement for transcription factor NFAT in interleukin-2 expression.
Mol. Cell. Biol.
19:2300-2307[Abstract/Free Full Text].
|
| 9.
|
Chuvpilo, S.,
A. Avots,
F. Berberich-Siebelt,
J. Glockner,
C. Fischer,
A. Kerstan,
C. Escher,
I. Inashkina,
F. Hlubek,
E. Jankevics,
T. Brabletz, and E. Serfling.
1999.
Multiple NF-ATc isoforms with individual transcriptional properties are synthesized in T lymphocytes.
J. Immunol.
162:7294-7301[Abstract/Free Full Text].
|
| 10.
|
Chuvpilo, S.,
M. Zimmer,
A. Kerstan,
J. Glockner,
A. Avots,
C. Escher,
C. Fischer,
I. Inashkina,
E. Jankevics,
F. Berberich-Siebelt,
E. Schmitt, and E. Serfling.
1999.
Alternative polyadenylation events contribute to the induction of NF-ATc in effector T cells.
Immunity
10:261-269[CrossRef][Medline].
|
| 11.
|
Clipstone, N. A., and G. R. Crabtree.
1992.
Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation.
Nature
357:695-697[CrossRef][Medline].
|
| 12.
|
Crabtree, G. R.
1999.
Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT.
Cell
96:611-614[CrossRef][Medline].
|
| 13.
|
Dickens, M.,
J. S. Rogers,
J. Cavanagh,
A. Raitano,
Z. Xia,
J. R. Halpern,
M. E. Greenberg,
C. L. Sawyers, and R. J. Davis.
1997.
A cytoplasmic inhibitor of the JNK signal transduction pathway.
Science
277:693-696[Abstract/Free Full Text].
|
| 14.
|
Dignam, J. D.,
P. L. Martin,
B. S. Shastry, and R. G. Roeder.
1983.
Eukaryotic gene transcription with purified components.
Methods Enzymol.
101:582-598[Medline].
|
| 15.
|
Dong, C.,
D. D. Yang,
M. Wysk,
A. J. Whitmarsh,
R. J. Davis, and R. A. Flavell.
1998.
Defective T cell differentiation in the absence of Jnk1.
Science
282:2092-2095[Abstract/Free Full Text].
|
| 16.
|
Durand, D. B.,
J. P. Shaw,
M. R. Bush,
R. E. Replogle,
R. Belagaje, and G. R. Crabtree.
1988.
Characterization of antigen receptor response elements within the interleukin-2 enhancer.
Mol. Cell. Biol.
8:1715-1724[Abstract/Free Full Text].
|
| 17.
|
Harlow, E., and D. Lane.
1988.
Antibodies: a laboratory manual.
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
|
| 18.
|
Hoey, T.,
Y. L. Sun,
K. Williamson, and X. Xu.
1995.
Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins.
Immunity
2:461-472[CrossRef][Medline].
|
| 19.
|
Ip, Y. T., and R. J. Davis.
1998.
Signal transduction by the c-Jun N-terminal kinase (JNK) from inflammation to development.
Curr. Opin. Cell Biol.
10:205-219[CrossRef][Medline].
|
| 20.
|
Jain, J.,
C. Loh, and A. Rao.
1995.
Transcriptional regulation of the IL-2 gene.
Curr. Opin. Immunol.
7:333-342[CrossRef][Medline].
|
| 21.
|
Jain, J.,
P. G. McCaffrey,
Z. Miner,
T. K. Kerppola,
J. N. Lambert,
G. L. Verdine,
T. Curran, and A. Rao.
1993.
The T-cell transcription factor NFATp is a substrate for calcineurin and interacts with Fos and Jun.
Nature
365:352-355[CrossRef][Medline].
|
| 22.
|
Kehlenbach, R. H.,
A. Dickmanns, and L. Gerace.
1998.
Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT in vitro.
J. Cell Biol.
141:863-874[Abstract/Free Full Text].
|
| 23.
|
Kuan, C. Y.,
D. D. Yang,
D. R. Samanta Roy,
R. J. Davis,
P. Rakic, and R. A. Flavell.
1999.
The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development.
Neuron
22:667-676[CrossRef][Medline].
|
| 24.
|
Li, B.,
C. Tournier,
R. J. Davis, and R. A. Flavell.
1999.
Regulation of IL-4 expression by the transcription factor JunB during T helper cell differentiation.
EMBO J.
18:420-432[CrossRef][Medline].
|
| 25.
|
Northrop, J. P.,
S. N. Ho,
L. Chen,
D. J. Thomas,
L. A. Timmerman,
G. P. Nolan,
A. Admon, and G. R. Crabtree.
1994.
NF-AT components define a family of transcription factors targeted in T-cell activation.
Nature
369:497-502[CrossRef][Medline].
|
| 26.
|
Park, J.,
A. Takeuchi, and S. Sharma.
1996.
Characterization of a new isoform of the NFAT (nuclear factor of activated T cells) gene family member NFATc.
J. Biol. Chem.
271:20914-20921[Abstract/Free Full Text].
|
| 27.
|
Pawson, T., and J. D. Scott.
1997.
Signaling through scaffold, anchoring, and adaptor proteins.
Science
278:2075-2080[Abstract/Free Full Text].
|
| 28.
|
Perrino, B. A.,
Y. L. Fong,
D. A. Brickey,
Y. Saitoh,
Y. Ushio,
K. Fukunaga,
E. Miyamoto, and T. R. Soderling.
1992.
Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.
J. Biol. Chem.
267:15965-15969[Abstract/Free Full Text].
|
| 29.
|
Porter, C. M.,
M. A. Havens, and N. A. Clipstone.
2000.
Identification of amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization.
J. Biol. Chem.
275:3543-3551[Abstract/Free Full Text].
|
| 30.
|
Ranger, A. M.,
M. R. Hodge,
E. M. Gravallese,
M. Oukka,
L. Davidson,
F. W. Alt,
F. C. de la Brousse,
T. Hoey,
M. Grusby, and L. H. Glimcher.
1998.
Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc.
Immunity
8:125-134[CrossRef][Medline].
|
| 31.
|
Rao, A.,
C. Luo, and P. G. Hogan.
1997.
Transcription factors of the NFAT family: regulation and function.
Annu. Rev. Immunol.
15:707-747[CrossRef][Medline].
|
| 32.
|
Sabapathy, K.,
Y. Hu,
T. Kallunki,
M. Schreiber,
J. P. David,
W. Jochum,
E. F. Wagner, and M. Karin.
1999.
JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development.
Curr. Biol.
9:116-125[CrossRef][Medline].
|
| 33.
|
Sherman, M. A.,
D. R. Powell,
D. L. Weiss, and M. A. Brown.
1999.
NF-ATc isoforms are differentially expressed and regulated in murine T and mast cells.
J. Immunol.
162:2820-2828[Abstract/Free Full Text].
|
| 34.
|
Shibasaki, F.,
E. R. Price,
D. Milan, and F. McKeon.
1996.
Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF-AT4.
Nature
382:370-373[CrossRef][Medline].
|
| 35.
|
Su, B.,
E. Jacinto,
M. Hibi,
T. Kallunki,
M. Karin, and Y. Ben-Neriah.
1994.
JNK is involved in signal integration during costimulation of T lymphocytes.
Cell
77:727-736[CrossRef][Medline].
|
| 36.
|
Szabo, S. J.,
J. S. Gold,
T. L. Murphy, and K. M. Murphy.
1993.
Identification of cis-acting regulatory elements controlling interleukin-4 gene expression in T cells: roles for NF-Y and NF-ATc.
Mol. Cell. Biol.
13:4793-4805[Abstract/Free Full Text]. (Erratum, 13:5928.)
|
| 37.
|
Timmerman, L. A.,
N. A. Clipstone,
S. N. Ho,
J. P. Northrop, and G. R. Crabtree.
1996.
Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression.
Nature
383:837-840[CrossRef][Medline].
|
| 38.
|
Weiss, L.,
A. J. Whitmarsh,
D. D. Yang,
M. Rincon,
R. J. Davis, and R. A. Flavell.
2000.
Signal transduction by activated MAP kinase regulated by selective expression in differentiated cells.
J. Exp. Med.
191:139-146[Abstract/Free Full Text].
|
| 39.
|
Whitmarsh, A. J.,
J. Cavanagh,
C. Tournier,
J. Yasuda, and R. J. Davis.
1998.
A mammalian scaffold complex that selectively mediates MAP kinase activation.
Science
281:1671-1674[Abstract/Free Full Text].
|
| 40.
|
Whitmarsh, A. J., and R. J. Davis.
1998.
Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals.
Trends Biochem. Sci.
23:481-485[CrossRef][Medline].
|
| 41.
|
Yang, D. D.,
D. Conze,
A. J. Whitmarsh,
T. Barrett,
R. J. Davis,
M. Rincon, and R. A. Flavell.
1998.
Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2.
Immunity
9:575-585[CrossRef][Medline].
|
| 42.
|
Yang, D. D.,
C. Y. Kuan,
A. J. Whitmarsh,
M. Rincon,
T. S. Zheng,
R. J. Davis,
P. Rakic, and R. A. Flavell.
1997.
Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene.
Nature
389:865-870[CrossRef][Medline].
|
| 43.
|
Yang, J.,
E. S. Bardes,
J. D. Moore,
J. Brennan,
M. A. Powers, and S. Kornbluth.
1998.
Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.
Genes Dev.
12:2131-2143[Abstract/Free Full Text].
|
| 44.
|
Yoshida, H.,
H. Nishina,
H. Takimoto,
L. E. Marengere,
A. C. Wakeham,
D. Bouchard,
Y. Y. Kong,
T. Ohteki,
A. Shahinian,
M. Bachmann,
P. S. Ohashi,
J. M. Penninger,
G. R. Crabtree, and T. W. Mak.
1998.
The transcription factor NF-ATc1 regulates lymphocyte proliferation and Th2 cytokine production.
Immunity
8:115-124[CrossRef][Medline].
|
| 45.
|
Zhu, J., and F. McKeon.
1999.
NF-AT activation requires suppression of Crm1-dependent export by calcineurin.
Nature
398:256-260[CrossRef][Medline].
|
| 46.
|
Zhu, J.,
F. Shibasaki,
R. Price,
J. C. Guillemot,
T. Yano,
V. Dotsch,
G. Wagner,
P. Ferrara, and F. McKeon.
1998.
Intramolecular masking of nuclear import signal on NF-AT4 by casein kinase I and MEKK1.
Cell
93:851-861[CrossRef][Medline].
|
Molecular and Cellular Biology, July 2000, p. 5227-5234, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Zhang, Y., Reynolds, J. M., Chang, S. H., Martin-Orozco, N., Chung, Y., Nurieva, R. I., Dong, C.
(2009). MKP-1 Is Necessary for T Cell Activation and Function. J. Biol. Chem.
284: 30815-30824
[Abstract]
[Full Text]
-
Yang, T. T. C., Yu, R. Y. L., Agadir, A., Gao, G.-J., Campos-Gonzalez, R., Tournier, C., Chow, C.-W.
(2008). Integration of Protein Kinases mTOR and Extracellular Signal-Regulated Kinase 5 in Regulating Nucleocytoplasmic Localization of NFATc4. Mol. Cell. Biol.
28: 3489-3501
[Abstract]
[Full Text]
-
Srinivasan, M., Frauwirth, K. A.
(2007). Reciprocal NFAT1 and NFAT2 Nuclear Localization in CD8+ Anergic T Cells Is Regulated by Suboptimal Calcium Signaling. J. Immunol.
179: 3734-3741
[Abstract]
[Full Text]
-
Bogoyevitch, M. A., Kobe, B.
(2006). Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases. Microbiol. Mol. Biol. Rev.
70: 1061-1095
[Abstract]
[Full Text]
-
Bronisz, A., Sharma, S. M., Hu, R., Godlewski, J., Tzivion, G., Mansky, K. C., Ostrowski, M. C.
(2006). Microphthalmia-associated Transcription Factor Interactions with 14-3-3 Modulate Differentiation of Committed Myeloid Precursors. Mol. Biol. Cell
17: 3897-3906
[Abstract]
[Full Text]
-
Proksch, P., Giaisi, M., Treiber, M. K., Palfi, K., Merling, A., Spring, H., Krammer, P. H., Li-Weber, M.
(2005). Rocaglamide Derivatives Are Immunosuppressive Phytochemicals That Target NF-AT Activity in T Cells. J. Immunol.
174: 7075-7084
[Abstract]
[Full Text]
-
Ortega-Perez, I., Cano, E., Were, F., Villar, M., Vazquez, J., Redondo, J. M.
(2005). c-Jun N-terminal Kinase (JNK) Positively Regulates NFATc2 Transactivation through Phosphorylation within the N-terminal Regulatory Domain. J. Biol. Chem.
280: 20867-20878
[Abstract]
[Full Text]
-
Yang, T. T. C., Xiong, Q., Graef, I. A., Crabtree, G. R., Chow, C.-W.
(2005). Recruitment of the Extracellular Signal-Regulated Kinase/Ribosomal S6 Kinase Signaling Pathway to the NFATc4 Transcription Activation Complex. Mol. Cell. Biol.
25: 907-920
[Abstract]
[Full Text]
-
Edmunds, J. W., Mahadevan, L. C.
(2004). MAP kinases as structural adaptors and enzymatic activators in transcription complexes. J. Cell Sci.
117: 3715-3723
[Abstract]
[Full Text]
-
Venkatesh, N., Feng, Y., DeDecker, B., Yacono, P., Golan, D., Mitchison, T., McKeon, F.
(2004). Chemical genetics to identify NFAT inhibitors: Potential of targeting calcium mobilization in immunosuppression. Proc. Natl. Acad. Sci. USA
101: 8969-8974
[Abstract]
[Full Text]
-
Zaichuk, T. A., Shroff, E. H., Emmanuel, R., Filleur, S., Nelius, T., Volpert, O. V.
(2004). Nuclear Factor of Activated T Cells Balances Angiogenesis Activation and Inhibition. JEM
199: 1513-1522
[Abstract]
[Full Text]
-
Diehl, S., Krahl, T., Rinaldi, L., Norton, R., Irvin, C. G., Rincon, M.
(2004). Inhibition of NFAT Specifically in T Cells Prevents Allergic Pulmonary Inflammation. J. Immunol.
172: 3597-3603
[Abstract]
[Full Text]
-
Gomez, M. F., Bosc, L. V. G., Stevenson, A. S., Wilkerson, M. K., Hill-Eubanks, D. C., Nelson, M. T.
(2003). Constitutively Elevated Nuclear Export Activity Opposes Ca2+-dependent NFATc3 Nuclear Accumulation in Vascular Smooth Muscle: ROLE OF JNK2 AND Crm-1. J. Biol. Chem.
278: 46847-46853
[Abstract]
[Full Text]
-
Wu, C.-C., Hsu, S.-C., Shih, H.-m., Lai, M.-Z.
(2003). Nuclear Factor of Activated T Cells c Is a Target of p38 Mitogen-Activated Protein Kinase in T Cells. Mol. Cell. Biol.
23: 6442-6454
[Abstract]
[Full Text]
-
Hogan, P. G., Chen, L., Nardone, J., Rao, A.
(2003). Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev.
17: 2205-2232
[Full Text]
-
Marin, O., Bustos, V. H., Cesaro, L., Meggio, F., Pagano, M. A., Antonelli, M., Allende, C. C., Pinna, L. A., Allende, J. E.
(2003). Inaugural Article: A noncanonical sequence phosphorylated by casein kinase 1 in {beta}-catenin may play a role in casein kinase 1 targeting of important signaling proteins. Proc. Natl. Acad. Sci. USA
100: 10193-10200
[Abstract]
[Full Text]
-
Dreikhausen, U. E., Gorf, K., Resch, K., Szamel, M.
(2003). Protein kinase C{beta}1, a major regulator of TCR-CD28-activated signal transduction leading to IL-2 gene transcription and secretion. Int Immunol
15: 1089-1098
[Abstract]
[Full Text]
-
Utsugi, M., Dobashi, K., Ishizuka, T., Endou, K., Hamuro, J., Murata, Y., Nakazawa, T., Mori, M.
(2003). c-Jun N-Terminal Kinase Negatively Regulates Lipopolysaccharide-Induced IL-12 Production in Human Macrophages: Role of Mitogen-Activated Protein Kinase in Glutathione Redox Regulation of IL-12 Production. J. Immunol.
171: 628-635
[Abstract]
[Full Text]
-
Didichenko, S. A., Fragoso, C. M., Thelen, M.
(2003). Mitotic and Stress-induced Phosphorylation of HsPI3K-C2{alpha} Targets the Protein for Degradation. J. Biol. Chem.
278: 26055-26064
[Abstract]
[Full Text]
-
Zipper, L. M., Mulcahy, R. T.
(2003). Erk Activation Is Required for Nrf2 Nuclear Localization during Pyrrolidine Dithiocarbamate Induction of Glutamate Cysteine Ligase Modulatory Gene Expression in HepG2 Cells. Toxicol Sci
73: 124-134
[Abstract]
[Full Text]
-
Yang, T. T. C., Chow, C.-W.
(2003). Transcription Cooperation by NFAT{middle dot}C/EBP Composite Enhancer Complex. J. Biol. Chem.
278: 15874-15885
[Abstract]
[Full Text]
-
Jorritsma, P. J., Brogdon, J. L., Bottomly, K.
(2003). Role of TCR-Induced Extracellular Signal-Regulated Kinase Activation in the Regulation of Early IL-4 Expression in Naive CD4+ T Cells. J. Immunol.
170: 2427-2434
[Abstract]
[Full Text]
-
Yang, Y.-M., Bost, F., Charbono, W., Dean, N., McKay, R., Rhim, J. S., Depatie, C., Mercola, D.
(2003). C-Jun NH2-terminal Kinase Mediates Proliferation and Tumor Growth of Human Prostate Carcinoma. Clin. Cancer Res.
9: 391-401
[Abstract]
[Full Text]
-
Sheridan, C. M., Heist, E. K., Beals, C. R., Crabtree, G. R., Gardner, P.
(2002). Protein Kinase A Negatively Modulates the Nuclear Accumulation of NF-ATc1 by Priming for Subsequent Phosphorylation by Glycogen Synthase Kinase-3. J. Biol. Chem.
277: 48664-48676
[Abstract]
[Full Text]
-
Gomez, M. F., Stevenson, A. S., Bonev, A. D., Hill-Eubanks, D. C., Nelson, M. T.
(2002). Opposing Actions of Inositol 1,4,5-Trisphosphate and Ryanodine Receptors on Nuclear Factor of Activated T-cells Regulation in Smooth Muscle. J. Biol. Chem.
277: 37756-37764
[Abstract]
[Full Text]
-
San-Antonio, B., Iniguez, M. A., Fresno, M.
(2002). Protein Kinase Czeta Phosphorylates Nuclear Factor of Activated T Cells and Regulates Its Transactivating Activity. J. Biol. Chem.
277: 27073-27080
[Abstract]
[Full Text]
-
Diehl, S., Chow, C.-W., Weiss, L., Palmetshofer, A., Twardzik, T., Rounds, L., Serfling, E., Davis, R. J., Anguita, J., Rincon, M.
(2002). Induction of NFATc2 Expression by Interleukin 6 Promotes T Helper Type 2 Differentiation. JEM
196: 39-49
[Abstract]
[Full Text]
-
Yang, T. T. C., Xiong, Q., Enslen, H., Davis, R. J., Chow, C.-W.
(2002). Phosphorylation of NFATc4 by p38 Mitogen-Activated Protein Kinases. Mol. Cell. Biol.
22: 3892-3904
[Abstract]
[Full Text]
-
Antos, C. L., McKinsey, T. A., Frey, N., Kutschke, W., McAnally, J., Shelton, J. M., Richardson, J. A., Hill, J. A., Olson, E. N.
(2002). Activated glycogen synthase-3beta suppresses cardiac hypertrophy in vivo. Proc. Natl. Acad. Sci. USA
10.1073/pnas.231619298v1
[Abstract]
[Full Text]
-
Buggins, A. G. S., Milojkovic, D., Arno, M. J., Lea, N. C., Mufti, G. J., Thomas, N. S. B., Hirst, W. J. R.
(2001). Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-{kappa}B, c-Myc, and pRb Pathways. J. Immunol.
167: 6021-6030
[Abstract]
[Full Text]
-
Yang, T., Davis, R. J., Chow, C.-W.
(2001). Requirement of Two NFATc4 Transactivation Domains for CBP Potentiation. J. Biol. Chem.
276: 39569-39576
[Abstract]
[Full Text]
-
Kyriakis, J. M., Avruch, J.
(2001). Mammalian Mitogen-Activated Protein Kinase Signal Transduction Pathways Activated by Stress and Inflammation. Physiol. Rev.
81: 807-869
[Abstract]
[Full Text]
-
Behrens, A., Sabapathy, K., Graef, I., Cleary, M., Crabtree, G. R., Wagner, E. F.
(2001). Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT). Proc. Natl. Acad. Sci. USA
98: 1769-1774
[Abstract]
[Full Text]
-
Haq, S., Choukroun, G., Lim, H., Tymitz, K. M., del Monte, F., Gwathmey, J., Grazette, L., Michael, A., Hajjar, R., Force, T., Molkentin, J. D.
(2001). Differential Activation of Signal Transduction Pathways in Human Hearts With Hypertrophy Versus Advanced Heart Failure. Circulation
103: 670-677
[Abstract]
[Full Text]
-
Haq, S., Choukroun, G., Kang, Z. B., Ranu, H., Matsui, T., Rosenzweig, A., Molkentin, J. D., Alessandrini, A., Woodgett, J., Hajjar, R., Michael, A., Force, T.
(2000). Glycogen Synthase Kinase-3{beta} Is a Negative Regulator of Cardiomyocyte Hypertrophy. JCB
151: 117-130
[Abstract]
[Full Text]
-
Constant, S. L., Dong, C., Yang, D. D., Wysk, M., Davis, R. J., Flavell, R. A.
(2000). JNK1 Is Required for T Cell-Mediated Immunity Against Leishmania major Infection. J. Immunol.
165: 2671-2676
[Abstract]
[Full Text]
-
Krause, D., Lyons, A., Fennelly, C., O'Connor, R.
(2001). Transient Activation of Jun N-terminal Kinases and Protection from Apoptosis by the Insulin-like Growth Factor I Receptor Can Be Suppressed by Dicumarol. J. Biol. Chem.
276: 19244-19252
[Abstract]
[Full Text]
-
Stevenson, A. S., Gomez, M. F., Hill-Eubanks, D. C., Nelson, M. T.
(2001). NFAT4 Movement in Native Smooth Muscle. A ROLE FOR DIFFERENTIAL Ca2+ SIGNALING. J. Biol. Chem.
276: 15018-15024
[Abstract]
[Full Text]
-
Antos, C. L., McKinsey, T. A., Frey, N., Kutschke, W., McAnally, J., Shelton, J. M., Richardson, J. A., Hill, J. A., Olson, E. N.
(2002). Activated glycogen synthase-3beta suppresses cardiac hypertrophy in vivo. Proc. Natl. Acad. Sci. USA
99: 907-912
[Abstract]
[Full Text]
-
Arbour, N., Naniche, D., Homann, D., Davis, R. J., Flavell, R. A., Oldstone, M. B.A.
(2002). c-Jun NH2-Terminal Kinase (JNK)1 and JNK2 Signaling Pathways Have Divergent Roles in CD8+ T Cell-mediated Antiviral Immunity. JEM
195: 801-810
[Abstract]
[Full Text]
Top
Abstract
Introduction
